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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Several mutagenicity studies have been conducted in in-vitro test systems where the Ames test (OECD 471) and the Mammalian Chromosomal Aberration Assay in CHO cells (OECD 473), turned out to be negative. This indicate clearly a non mutagenic potential in in-vitro conditions.

Altogether, it was judged that FAT 40871/A TE substance is non mutagenic in vitro.


Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: 29 September 2015 and Experimental Completion Date: 25 October 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP Guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to basepair substitution mutations, rather than frameshift mutations (Green and Muriel, 1976).
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 and hamster liver S9 was used as the metabolic activation system.
Test concentrations with justification for top dose:
- Initial toxicity-mutation assay (Experiment B1), the dose levels tested were: 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate.
- In the confirmatory mutagenicity assay (experiment B2), the dose levels tested were: 50.0, 150, 500, 1500, 5000 μg per plate.

To achieve a solution, the most concentrated dilution was vortexed for 3 minutes and sonicated at 35.1 to 35.9ºC for 10 to 13 minutes in each assay. Test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under filtered light.
Vehicle / solvent:
Water was used as the vehicle.

- Vehicle: Water
- CAS Number: 7732-18-5
- Supplier: Sigma-Aldrich
- Lot Number: RNBD5189/RNBD7780
- Purity: Sterile-filtered
- Expiration Date: November 2016/April 2017
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
2-AA: Positive control used for test performed with metabolic activation S9 from rat liver (oxidative). Used at 1.0 μg/plate for TA98 and TA1535. Used at 2.0 μg/plate for starin TA100 and 1537. Used at 15 μg/plate with starin WP2 uvrA.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Water
True negative controls:
no
Positive controls:
yes
Positive control substance:
congo red
Remarks:
Positive control used for test performed with metabolic activation S9 from hamster liver (reductive).
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Water
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Positive control used at 1.0 ug/plate, without matebolic activation for strain TA98 .
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Positive control used at 1.0 umg/plate without metabolic activation for strain TA100 and TA1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Water
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Posotive control used at 75 ug/plate without metabolic activation for strain TA1537.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Water
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Positive control used at 1000 ug/plate without metabolic activation for starin WP2 uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: Pre-incubation method.

CONDITIONS OF PRE-CULTURE:
Time of pre-culture: Approxymately 12 hours
Culture vessel (shape and volume): cylinder, 150 ml
Volume of culture medium: 30 to 50 mL
Amount of starin inoculated: 1 colony or around 100 uL


PRE-INCUBATION:
Temperature: 37°C (for without S9 and with oxidative S9)
30°C (for reductive S9)

Time: 60 minutes (for without S9 and with oxidative S9)
30 minutes (for reductive S9)

INCUBATION:
Temperature: 37°C
Time: 48 to 72 hours

NUMBER OF REPLICATIONS:
- Initial toxicity-mutation assay (experiment B1): 2 replicates
- In the confirmatory mutagenicity assay (experiment B2): 3 replicates

COLONY COUNTING METHOD: Automatic counting
Evaluation criteria:
Evaluation of Test Results
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance as specified below:

Strains TA1535 and TA1537
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value.

Strains TA98, TA100 and WP2 uvrA
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value.

An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was neither positive nor equivocal.
Statistics:
Not avaialble
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Sterility Results
No contaminant colonies were observed on the sterility plates for the vehicle control, the test substance dilutions or the S9 and Sham mixes.


Tester strain titer results:
Tester Strain
TA98 TA100 TA1535 TA1537 WP2 uvrA
Experiment Titer Value (x 109 cells per mL)
B1 2.1 1.4 1.4 1.8 1.7
B2 2.4 2.0 1.4 1.9 2.8


Initial Toxicity-Mutation Assay:
The results of the preliminary toxicity assay conducted at dose levels of 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate in water.
The maximum dose of 5000 μg per plate was achieved using a concentration of 50.0 mg/mL and a 100 μL plating aliquot. Neither precipitate nor toxicity was observed.

Confirmatory Mutagenicity Assay
Based upon the results of the preliminary toxicity assay, the dose levels selected for the mutagenicity assay were 50.0, 150, 500, 1500, 5000 μg per plate. Neither precipitate nor toxicity was observed. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.


CONCLUSION
All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, FAT 40871/A TE did not cause a positive mutagenic response with any of the tester strains in either the absence of S9 activation or in the presence of oxidative or reductive S9 activation.


Remarks on result:
other: Neither precipitate nor toxicity was observed

Historical Negative and Positive Control Values 2014 Revertants per plate

                                 Activation
                  None              Rat Liver
 Strain  Control  Mean SD  Min  Max  95% CL  Mean  SD  Min  Max  95% CL 
 TA98  Neg  16 42   6 -26  24 53  10 -38 
 TA98  Pos  232 258  57  2691     400 165  109  1382   
 TA100  Neg  94 14  66  152   66 -122  102 18  63  164  66 -138 
 TA100  Pos  681 176  213  1767    681 259  186  2793   
 TA1535  Neg  11 31  3 -19   13 36  3 -23 
 TA1535  Pos  586 226  16  2509     117 99  23  1060   
 TA1537  Neg  7 19   1 -13  9 23  1 -17 
 TA1537  Pos  411 355  32  2921     72 52  10  562   
 WP2 uvrA  Neg  25 7 7  62    11 -39  28 10  55  12 -44 
 WP2 uvrA  Pos  376 123  99  1026     302 102  91  687   
SD=standard deviation; Min=minimum value; Max=maximum value; 95% CL = Mean ±2 SD (but not less than zero); Neg=negative control (including but not limited to deionized water, dimethyl sulfoxide, ethanol and acetone); Pos=positive control                            

Historical Negative and Positive Control Values Hamster Liver S9 (2011-2014) revertants per plate

ACTIVATION/HAMSTER LIVER

 Strain  Control  Mean SD  Min  Max  95% CL 
 TA98  Neg  32 13 47 16 -48
 TA98  Pos  304 389  93  1711   
 TA100  Neg  94 14  72  127  66 -122 
 TA100  Pos  1498 868  597  2727   
 TA1535  Neg  14 27  4 -24 
 TA1535  Pos  398 107  269  589   
 TA1537  Neg  14  5  19  6 -22
 TA1537  Pos  487 123  328  743   
 WP2 uvrA  Neg 25  9  10  42  7 -43
 WP2 uvrA  Pos  301 34  267  345   
SD=standard deviation; Min=minimum value; Max=maximum value; 95% CL = Mean ±2 SD (but not less than zero); Neg=negative control (including but not limited to deionized water, dimethyl sulfoxide, ethanol and acetone); Pos=positive control                
Conclusions:
All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, FAT 40871/A TE did not cause a positive mutagenic response with any of the tester strains in either the absence of S9 activation or in the presence of oxidative or reductive S9 activation.
Executive summary:

The test substance, was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the absence of S9 activation and in the presence of both Aroclor-induced rat liver S9 activation (oxidative) and uninduced hamster liver S9 activation (reductive). Water was used as the vehicle.

In the initial toxicity-mutation assay, the dose levels tested were 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. Neither precipitate nor toxicity was observed. Based upon these results, the maximum dose tested in the mutagenicity assay was 5000 μg per plate.

In the confirmatory mutagenicity assay, the dose levels tested were 50.0, 150, 500, 1500, 5000 μg per plate. Neither precipitate nor toxicity was observed. No positive mutagenic responses were observed with any of the tester strains in either the absence of S9 activation or in the presence of oxidative or reductive S9 activation.

These results indicate wthat the test item was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in either the absence of S9 activation or in the presence of oxidative or reductive S9 activation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A reliable bacterial reverse mutation test and an in vitro chromosome aberration in Chinese Hamster Ovary Cells test, are available and were all performed according to OECD/EC guidelines.

Regarding the both in vitro test, the Ames test turnes to be negative together with the chromosome aberration assay in CHO cells.
All these both studies are taken into consideration.

FAT 40871/A TE was found to be be negative in the Ames test (OECD 471) and also negative in the chromosme aberration in CHO cells test (OECD 473).


Justification for classification or non-classification

Based on the above mentioned results the substance does not need to be classified according to CLP regulation (Regulation EC No. 1272/2008) and DSD (Directive 67/548/EEC).